Nitric Oxide Colorimetric Detection Kit

Cat# OKAU00021

Size : 2plate

Brand : Aviva Systems Biology

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Datasheets/ManualsPrintable datasheet for Nitric Oxide Colorimetric Detection Kit (OKAU00021)
Product Info
ELISA Kit Detection MethodColorimetric
ELISA Kit LinearityLinearity was determined by taking two diluted human urine samples with known Nitrite and Total NO Concentrations and mixing them in the ratios given below. The measured concentrations were compared to the expected values based on the ratios used.
High Urine SampleLow Urine SampleExpected Conc. (uM)Observed Conc. (uM)% Recovery
NitriteTotal NONitriteTotal NONitriteTotal NO
80%20%61.6122.862.5121.0101.5%98.5%
60%40%51.9111.052.7110.7101.5%99.7%
40%60%42.299.242.895.6101.6%96.4%
20%80%32.487.332.284.799.5%97.0%
Mean Recovery101.0%97.9%
ELISA Kit PrincipleThe DetectX® Nitric Oxide Detection Kit is designed to quantitatively measure Nitrate and Nitrite present in a variety of samples. Nitric Oxide content is derived from the sum of Nitrate (- NO3 ) and Nitrite (- NO2 ). Please read the complete kit insert before performing this assay. Both Nitrate and Nitrite standards are provided to generate standard curves for the assay and all samples should be read off the appropriate standard curve. For Nitrite detection, samples are mixed with Color Reagents A and B and incubated at room temperature for 5 minutes. The colored product is read at 540 - 570 nm. The concentration of Nitrite in the sample is calculated, after making a suitable correction for any dilution of the sample, using software available with most plate readers. Total Nitric Oxide content is measured after the sample is incubated with Nitrate Reductase and NADH. The reductase in combination with NADH reduces Nitrate to Nitrite. After a 20 minute incubation at room temperature, Color Reagents A and B are added and incubated at room temperature for 5 minutes. The colored product is read and calculated as with the Nitrite determination above. The concentration of Nitrate in the sample is calculated by subtracting the measured Nitrite concentration from the Total Nitric Oxide concentration for the sample. This kit uses Nitrate and Nitrite Standard solutions calibrated to the US National Institute for Science and Technology Standard Reference Materials and ISO/IEC standards.
ELISA Kit ReproducibilityIntra Assay Precision Three samples were diluted in Assay Buffer and run in replicates of 20 in an assay. The mean and precision of the calculated Nitrite or Total NO concentrations were:
SampleNitrite Conc. (uM)%CVTotal NO Conc. (uM)%CV
145.14.470.56.8
273.39.1107.44.4
3132.71.3157.81.8
Inter Assay Precision Three samples were diluted in Assay Buffer and run in duplicates in twenty assays run over multiple days by three operators. The mean and precision of the calculated Nitrite or Total NO concentrations were:
SampleNitrite Conc. (uM)%CVTotal NO Conc. (uM)%CV
144.13.168.87.4
266.44.0112.15.7
3126.76.3154.44.1
ELISA Kit Component
ComponentQuantity
Clear Half Area 96 well Plates2 Plates
Nitrate Standard200 uL
Nitrite Standard200 uL
Assay Buffer60 mL
NADH Concentrate1.2 mL
Nitrate Reductase1 Vial
Enzyme Stabilization Buffer1 mL
Color Reagent A5 mL
Color Reagent B5 mL
Additional InformationBackground: Nitric oxide (NO) is a diffusible, transient, reactive molecule that has physiological effects in the picomolarto-micromolar range. Acting through soluble guanylate cyclase activation, NO is an important physiological regulator of the cardiovascular, nervous, and immunological systems. NO is bio-available by two routes. It can be endogenously generated by constitutive or induced enzymes like Nitric Oxide Synthase or it can be orally ingested as nitrates and nitrites for rapid uptake into circulation and subsequent conversion. The reactive nature of nitric oxide allows it to act as a cytotoxic factor when released during an immune response by cells such as macrophages. It also allows NO to be easily converted to a toxic radical that can produce nitrosative damage to cells, organelles and molecules such as DNA. Nitrosaylation is also a regulated post-translational modification in cell signaling. The balance and dynamics of the regulatory/ damage facets of NO are major forces in mitochondrial signaling and dysfunction. NO is linked not only to coronary heart disease, endothelial dysfunctions, erectile dysfunction, and neurological disorders, but also diabetes, chronic periodontitis, autism, cancer, and assorted age-related diseases. The physical properties of Nitric Oxide make it challenging for direct detection methods. However, colorimetric methods can be applied to measure its stable break-down products, nitrate (-NO3) and nitrite (-NO2).
::Detection Limit: The Limit of Detection was determined as 0.94 uM in the Nitirite and 3.0 uM in the Total Nitric Oxide assays.
Reconstitution and Storage2°C to 8°C
Sample TypeWater, Buffers, Serum, Plasma, Urine, Saliva, and TCM
SensitivitySensitivity was determined as 2.63 uM in the Nitrite and 1.02 uM in the Total Nitric Oxide assays.
Gene Full NameNitric Oxide Colorimetric